A broken tool and broken hearts

The analysis of the human genome showed that our DNA contains fewer genes than most scientists expected – and that most are more like archipelagoes than compact islands. A gene’s sequence usually contains several protein-encoding segments, called exons, which are separated by long, non-coding sequences called introns. These have to be removed to create a messenger RNA with a seamless protein-coding region. The job is handled by an assembly of molecules called the spliceosome, which often removes some of the exons as well. As a result, cells build versions of a protein with different modules and functions that are specific to certain tissues. This process of splicing turns proteins into multifunctional tools – like replacing a box full of screwdrivers with several bits that can be inserted into one handle. As a result, a complex organism can be built with a smaller toolbox.

Defects in splicing may cause the loss of a form of a protein and thus a tool that is essential in a particular tissue. This happens in a number of serious genetic diseases. For several years Norbert Hübner and Michael Gotthardt have believed that the list includes some forms of a heart disease called dilated cardiomyopathy (DCM). This condition is marked by an enlarged, weakened heart and is responsible for about a third of deaths from congestive heart failure. Now Norbert’s group, working with the labs of Michael, Nikolaus Rajewsky, Matthias Selbach and Markus Landthaler at the MDC, have shown that mutations in a molecule called RBM20 probably contribute to DCM by affecting the splicing of crucial molecules in the heart. Their work was reported in the Aug. 1 edition of the Journal of Clinical Investigation.

“Defects in splicing can have several causes,” Norbert says. “The spliceosome has to recognize the boundaries between introns and exons in the target RNA and remove the right sequence. That might not happen if the sequence of an RNA has been altered by a mutation in its gene. But the cause might also lie with a defect in the splicing machinery itself. The spliceosome is built from different components in various tissues, to handle their particular needs. If one of those components is defective, you might see a tissue-specific problem in splicing.”

Mutations in RBM20 had already been linked to dilated cardiomyopathy. The protein binds to RNAs, which suggested that it might participate in splicing. It is produced in high quantities in the heart and striated muscle, hinting that it had specific functions in these tissues. In 2012 Michael and Norbert and their colleagues showed that it helps regulate the splicing of an enormous protein called titin, which has crucial functions in muscle tissue. Titin acts as a sort of spring as piston-like muscle cells expand and contract. Improper splicing can create a spring that is too short or too long, changing the mechanical properties of muscle. In the heart this can lead to disaster.

Now the scientists have identified other targets of RBM20 and provided a detailed account of its operation. Their work helps explain how defects in the protein – or its targets – may cause the improper splicing of molecules that are crucial to the functions of the heart.

Answering such questions in the cell requires understanding how proteins fit together into a molecular machine such as the spliceosome and how and where they dock onto targets such as RNAs. Proteins’ shapes provide a physical and chemical architecture that permits them to interact with other molecules that have compatible structures. RBM20 was likely able to “read” a small pattern in RNAs that told the machine where to dock on.

“Our problem was that titin was the only known target of this protein,” says Henrike Maatz, a postdoc in Norbert’s group who headed the current study. “You can’t generalize a sequence pattern from only one example, so we needed to find other RNAs that it could bind to.”

The scientists used several approaches to obtain a list. One involved “gluing” the protein onto RNA molecules using a method called PAR-CLIP, provided by Markus Landthaler, extracting RBM20 from cells, and identifying the RNAs attached to it by sequencing them. They removed regions of the RNAs not directly bound to RBM20, leaving very short segments that contained the motif that allowed the RNAs to bind. This allowed Marvin Jens from Nikolaus’ group to search for a common pattern that RBM20 could detect. It turned out to be very short – only four nucleotide “letters” long.

Another strategy compared the complete set of RNAs produced by rats with healthy RBM20, versus those produced in a strain of rats that lacked the protein, and then comparing those to the molecules found in humans who had suffered heart failure.

Mutations in RBM20 might be affecting the spliceosome’s ability to recognize targets, but they could also be interfering with the operation of the machine itself. To get a closer look at its functions in the spliceosome, the scientists turned to Marie Kirchner from Matthias Selbach’s lab, which specializes in interactions between proteins. Marie provided important information about RBM20’s position in the machine, the other proteins in the spliceosome, and how it contributed to splicing. They also demonstrated that mutations found in some heart disease patients affected its ability to interact with other parts of the machine.

Putting all of this information together allowed the scientists to identify a number of new RNA targets. Studying RBM20’s target pattern revealed that it was located in introns. By docking onto this position, its normal function is to tell the spliceosome to remove a nearby exon, shortening the molecule. But in animals and people with a defective version of the protein, the exon is left in. This creates RNAs with extra segments that are subsequently used to build versions of essential proteins that are too long. In the case of titin, this creates a spring that is too slack and muscles that don’t contract efficiently. Ultimately the heart must work harder and becomes enlarged.

“The study has put a number of earlier findings into perspective,” Norbert says. “Mutations in several other molecules had been associated with dilated cardiomyopathies, but we didn’t understand how they contributed to the disease. Now we know that most of those molecules turn out to be targets of RBM20, possibly suffering defects in the way they are spliced. And we found several more targets that are probably similarly affected, but had not previously been linked to the condition. All of this gives us many more approaches toward understanding a complex and deadly disease.”

Featured Image: Mutations in a molecule called RBM20 probably contribute to dilated cardiomyopathy. Photo: Huebner lab, MDC